Therapeutic and diagnostic strategies

ABSTRACT

The present invention encompasses the finding that microRNAs (miRNAs) regulate certain key proteins involved in DNA repair. In some embodiments, a miRNA suppresses levels and/or activity of one or more DNA repair proteins. In some such embodiments, such suppression renders cells hypersensitive to certain DNA damage agents (e.g., γ-irradiation and genotoxic drugs, among others). The present invention provides various reagents and methods associated with these findings including, among other things, strategies for treating cell proliferative disorders, certain diagnostic systems, etc.

RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 13/063,155,filed Oct. 27, 2011, allowed, which claims the benefit under 35 U.S.C.§371 of international application serial number PCT/US2009/057506, filedSep. 18, 2009, which claims priority under 35 U.S.C. §119 to U.S. Ser.No. 61/098,696, filed on Sep. 19, 2008, entitled “miRNA Targets”, andU.S. Ser. No. 61/098,707, filed on Sep. 19, 2008, entitled “Therapeuticand Diagnostic Strategies”, the entire contents of which areincorporated herein by reference.

BACKGROUND

DNA repair mechanisms are central to a variety of cellular processes,including, importantly, DNA replication and cell proliferation. Abilityto influence DNA repair systems, and/or cell cycle progressiongenerally, can provide novel therapeutic and diagnostic approaches to avariety of diseases, disorders, and conditions associated with cellproliferation (including, for example, cancer, immune-mediated disordersand/or neurodegenerative disorders).

SUMMARY

The present invention encompasses the finding that microRNAs (miRNAs)regulate certain key proteins involved in DNA repair and/or cell cycleprogression. In some embodiments, a miRNA modulates levels and/oractivity of one or more DNA repair and/or cell cycle progressionproteins. In some such embodiments, such modulation renders cellshypersensitive to certain DNA damage agents (e.g., γ-irradiation andgenotoxic drugs, among others).

The present invention specifically encompasses the finding that certainmiRNAs whose expression is increased in terminally differentiated cellsregulate DNA repair and/or cell cycle progression proteins. In someembodiments, the miRNAs show increased expression in terminallydifferentiated hematopoietic cells. In some embodiments, the miRNAs areselected from the group consisting of miR-22, miR-125a, miR-24 (e.g.,miR-24-1; miR-24-2), miR-23 (e.g., miR-23a, miR-23B), miR-27 (e.g.,miR-27a, miR-27b), miR-17, miR-18, miR-19, miR-20, miR-34 a, miR-92,miR-125, miR-146 a, miR-155, miR-181 a, 200a, miR-48, miR-84, andmiR-241. In some embodiments, the miRNA is miR-24.

The present invention provides, among other things, systems formodulating DNA repair and/or cell cycle progression in cells by alteringexpression and/or activity of one or more such miRNAs.

The present invention provides, among other things, systems for reducingcell proliferation and/or increasing sensitivity to certain DNA damageagents through modulation of certain miRNAs and/or of miRNA-basedregulation of DNA repair proteins. According to the present invention,such systems are particularly useful, for example, in reducingundesirable cell proliferation (e.g., in the context of cancer,transplant rejection, T cell immunity, etc.). In some embodiments, cellswhose proliferation is to be reduced include hematopoietic cells. Insome embodiments, inventive systems for reducing cell proliferationinclude increasing levels and/or activity of certain miRNAs in cells,such that DNA repair is reduced and/or expression and/or activity of oneor more DNA repair (e.g., H2AX) or cell cycle progression proteins isaltered. In some such embodiments, cells may also be exposed to one ormore DNA damage agents (e.g., γ-irradiation and genotoxic drugs, amongothers). In some embodiments, the present invention provides systems forinducing apoptosis.

The present invention provides, among other things, systems forincreasing cell proliferation and/or decreasing sensitivity to certainDNA damage agents through modulation of certain miRNAs and/or ofmiRNA-based regulation of DNA repair and/or cell cycle progressionproteins. According to the present invention, such systems areparticularly useful, for example, in cell culture applications and/or inapplications where cell proliferation is desirably increased. In someembodiments, cells whose proliferation is to be increased includehematopoietic cells. In some embodiments, inventive systems forincreasing cell proliferation include decreasing levels and/or activityof certain miRNAs in cells such that DNA repair is reduced and/orexpression and/or activity of one or more DNA repair (e.g., H2AX) and/orcell cycle progression proteins is altered.

The present invention provides, among other things, systems fordetecting cells undergoing terminal differentiation and/or cells whoseDNA repair systems are compromised. Particularly useful applications ofsuch systems, according to the present invention include, among otherthings, diagnostics for identifying cells and/or individuals that areparticularly susceptible to DNA damage agents (e.g., γ-irradiation andgenotoxic drugs, among others).

DESCRIPTION OF THE DRAWING

FIG. 1A to FIG. 1D show kinetics of miR-24 and H2AX transcript levels inTPA-treated K562 and HL60 cells. TPA treatment of K562 cells (FIGS. 1A,1C) and HL60 cells (FIGS. 1B, 1D) increases miR-24 levels (FIGS. 1A, 1B)with a concurrent decrease in H2AX mRNA (FIGS. 1C, 1D).

FIG. 2A and FIG. 2B demonstrate, in FIG. 2A, that miR-328 does nottarget the 3′UTR of I-12AX mRNA in a luciferase reporter assay, whereHepG2 cells were transfected with control miRNA (black) or syntheticmiR-328 (white) for 48 hr and then with H2AX 3′UTR-luciferase reporter(H2AX) or vector (V) for 24 hr; and, in FIG. 2B, that miR-328over-expression in K562 cells has no effect on H2AX mRNA (left),analyzed by qRT-PCR normalized to GAPDH, and protein (right) 48 hrlater.

FIG. 3A to FIG. 3C show that chromosomal aberrations after γ-irradiationare greater in K562 than HepG2 cells. HepG2 cells express less miR-24(FIG. 3A, right panel) and more H2AX (FIG. 3A, left panel) than K562cells. Cells were either untreated or irradiated and incubated for 24 hbefore metaphase spreads were prepared. For each condition, at least 50metaphase spreads were examined. The average number of cells withchromosomal aberrations (FIG. 3B) and chromosomal aberrations per cell(FIG. 3C) were analyzed. In (FIG. 3B) and (FIG. 3C) white bars representK562 cells; black bars, HepG2 cells.

FIG. 4A to FIG. 4K show that miR-24 down-regulates H2AX expressionduring terminal differentiation. miR-24, analyzed by qRT-PCR relative toU6, increases during differentiation of K562 cells (FIG. 4A) with TPA tomegakaryocytes or hemin to erythrocytes (#, p<0.001, ***, p<0.005) andduring differentiation of HL60 cells (FIG. 4B) with TPA to macrophagesor DMSO to granulocytes (#, p<0.001). HL60 cells were treated with TPAfor 2 days (left panel) or DMSO for 5 days (right panel) and miR-24levels analyzed as described above. Under the same differentiatingconditions for K562 (FIG. 4C) and HL60 (FIG. 4D) cells, H2AX mRNA,normalized to GAPDH mRNA, is down-regulated (**, p<0.01, K562; ***,p<0.005, HL60). FIG. 4E shows that H2AX protein decreases after 2 d ofTPA differentiation. Relative H2AX expression was quantified bydensitometry using H3 as control. FIG. 4F shows that H2AX mRNA isselectively pulled down from K562 cell lysates with biotinylated miR-24(white) compared to control cel-miR-67 (black). For each condition,pulled down RNA was first normalized to GAPDH mRNA in the sample andthen to relative input cellular RNA (***, p<0.005). The housekeepinggene UBC was not enriched in the pull down. FIG. 4G shows a schematicrepresenting the location of miR-24 binding sites in the 3′UTR of H2AXmRNA. FIG. 4H shows that miR-24 targets the 3′UTR of H2AX mRNA in aluciferase reporter assay. HepG2 cells were transfected with controlmiRNA (black) or synthetic miR-24 (white) for 48 hr and then with H2AX3′UTR-luciferase reporter (H2AX) or vector (V) for 24 hr. Mean+SD,normalized to vector control, of 3 independent experiments are shown (*,p<0.001). miR-24 over-expression in HepG2 cells decreases H2AX mRNA,analyzed by qRT-PCR normalized to GAPDH (FIG. 4I; white, miR-24; black,cel-miR-67) and protein (FIG. 4J) 48 hr later. miR-24 over-expressiondoes not alter UBC mRNA levels. In (FIG. 4A to FIG. 4D; FIG. 4F; andFIGS. 4H and 4I), mean±SD are shown. FIG. 4K shows suppression of theluciferase activity of a reporter gene containing in its 3′ UTR the twopredicted miR-24 MRE, either wild-type (wt) or with mutated seed regions(mt). HepG2 cells were transfected with control miRNA (black) or miR-24mimic (white) for 48 h and then with the indicated H2AX 3′UTR-luciferase reporters or vector (V). Luciferase activity was assayed24 h later. Mean±s.d., normalized to vector control, of threeindependent experiments is shown. In all panels, mean s.d. is shown.

FIG. 5A to FIG. 5D show miR-24 expression impedes DSB repair and induceschromosomal instability of γ-irradiated K562 cells. FIG. 5A shows thattransfection of miR-24 mimic into K562 cells reduces H2AX comparably toTPA differentiation. H2AX was quantified relative to H3 protein bydensitometry. FIG. 5B shows representative images of metaphasechromosome spreads were prepared from treated cells 24 h afterγ-irradiation. Arrows mark chromosome breaks or fragments. FIG. 5C showschromosome breaks were quantified 24 hr after irradiation of K562 cellsthat were either undifferentiated (white) or had been differentiatedwith TPA (black, left panel) or transfected with miR-24 (black, middlepanel). In the right panel, differences in chromosome breaks that werenot present 24 hr following 0.375 Gy were significantly different 48 hrafter irradiation in miR-24 (black) vs mock-transfected (white) cells.The mean+SD number of chromosome breaks and fragments per cell,normalized to control is plotted. FIG. 5D shows that overexpressingmiR-24 increases unrepaired DSB by comet assay. K562 cells, transfectedwith miR-24 mimic and/or miR-24-insensitive H2AX expression plasmid,were treated or not with bleomycin (0.5 μg/ml) for 12 h and analyzed bysingle cell gel electrophoresis (comet assay) 12 h later. H2AX proteinis compared to H3 level in the immunoblot. H2AX levels, reduced by themiR-24 mimic, are rescued by the H2AX expression plasmid. Representativeimages from bleomycin-treated cells are shown in the upper panel and themean±SD comet tail moment for each condition below (black, controlmimic, expression vector; dark stippled bars, miR-24 mimic, vector;white, control mimic, H2AX expression plasmid; light stippled bars,miR-24 mimic, H2AX expression plasmid). Manipulating miR-24 or H2AXlevels did not affect baseline DNA damage, but DNA damage afterirradiation was significantly increased (p<0.001) in miR-24mimic-transfected cells, but only in the absence of H2AX rescue.

FIG. 6A to FIG. 6D show that cells overexpressing miR-24 arehypersensitive to DNA damage by cytotoxic drugs. FIG. 6A shows that K562cells overexpressing miR-24 (left) or treated with TPA (right) arehypersensitive to bleomycin, assessed by viability relative tomock-treated cells (□) 2 d later. TPA treatment (u) or transfection withmiR-24 mimic (m), but not miR-328 mimic (FIG. 6A), significantlysensitizes K562 cells to DNA damage (p<0.005). FIG. 6B shows similarlythat HepG2 cells overexpressing miR-24 (▪) are hypersensitive, comparedto mock-transfected cells (□) to bleomycin (left) and cisplatin (right).miR-24 over-expression significantly reduces viability to both genotoxicagents (p<0.004). miR-24-mediated hypersensitivity of K562 (FIG. 6C) andHepG2 (FIG. 6D) cells is rescued by expression of miR-24-insensitiveH2AX. Cells were mock transfected (El) or transfected with miR-24 mimic(u) or H2AX cDNA lacking the 3′UTR (FIG. 6A) or both (•). Cell viabilitywas assayed 2 d after exposure to DNA damage and depicted relative tothat of undamaged cells. Immunoblots demonstrate miR-24-mediateddecrease in H2AX protein and rescue by transfecting the H2AX cDNA (no3′UTR).

FIG. 7A to FIG. 7D shows that antagonizing miR-24 enhances cellresistance to bleomycin. FIG. 7A shows that miR-24 knockdown in K562cells, treated or not with TPA, specifically decreases miR-24 levels,assayed by qRT-PCR in cells transfected with miR-24 ASO relative tocontrol ASO (Ctl). miR-24 expression in ASO-treated cells, relative toU6 snRNA, is normalized to that in control ASO-treated cells. FIG. 7Bshows that miR-24 ASO enhances H2AX transcript (left) and protein levels(right) in K562 cells treated with TPA, but not in untreated, K562cells. FIG. 7C shows that relative to untreated cells (□), TPA treatment(•) sensitizes K562 cells to bleomycin treatment. Transfection of miR-24ASO significantly blocks bleomycin-induced apoptosis of TPA-treated K562cells (•, p<0.003), but does not affect apoptosis of untreated K562cells (FIG. 7A). FIG. 7D shows that transfection of miR-24 significantlyenhances repair of bleomycin-induced DNA damage, as measured by cometassay, in TPA-treated K562 cells (*, P o 0.001). Representative imagesfrom bleomycin-treated cells are shown on the left and the mean±s.d.comet tail moments of three independent experiments are shown on theright.

FIG. 8A to FIG. 8C shows that inhibition of miR-24 leads to increasedcell proliferation, while miR-24 over-expression leads to cell-cyclearrest. FIG. 8A shows that miR-24 knockdown in K562 cells specificallydecreases miR-24 levels, assayed by qRT-PCR in cells transfected withmiR-24 ASO (white) relative to control ASO (black). Expression relativeto U6 snRNA is depicted normalized to control cells. FIG. 8B shows thatmiR-24 knockdown with ASO increases K562 cell proliferation measured bythymidine uptake, both in the presence and absence of TPA. The declinein proliferation with TPA is completely restored by antagonizing miR-24.FIG. 8C shows that miR-24 over-expression increases the G1 compartmentin HepG2 cells. HepG2 cells transfected with miR-24 or control mimic for48 hr were stained with propidium iodide and analyzed by flow cytometry.Representative analysis of three independent experiments is shown. Errorbars represent standard deviation from 3 independent experiments (FIG.8A to FIG. 8C). *, p<0.05; ″, p<0.01; #, p<0.005.

FIG. 9A and FIG. 9B show, respectively, H2AX mRNA and kinetics ofthymidine incorporation in TPA-treated K562 cells. FIG. 9A shows H2AXmRNA analyzed by qRT-PCR using coding region primers decreases ˜4-foldduring TPA induced differentiation of K562 cells. These primers amplifyboth H2AX transcripts. GAPDH mRNA was used for normalization. FIG. 9Bshows kinetics of thymidine incorporation in TPA-treated K562 cells. By12 h there is no thymidine incorporation, indicating that cells havestopped dividing.

FIG. 10A and FIG. 10B show that miR-24 levels and H2AX levels in primaryhuman peripheral blood macrophages and granulocytes are comparable tocells generated by in vitro differentiation. FIG. 10A shows that miR-24,analyzed by qRT-PCR relative to U6, increases during TPA-induceddifferentiation of HL60 cells to macrophages, and this increased miR-24expression is also observed in primary human peripheral bloodmacrophages and granulocytes. FIG. 10B shows that H2AX mRNA (normalizedto UBC mRNA), and protein (normalized to histone H3) is down-regulatedduring differentiation of HL60 cells and is comparable to levels inprimary human peripheral blood macrophages and granulocytes.

FIG. 11 shows that representative images of metaphase chromosome spreadswere prepared from treated cells 24 h after γ-irradiation. Arrows markchromosome breaks or fragments.

FIG. 12 shows that miR-24-mediated hypersensitivity of K562 cells tobleomycin is not rescued by expression of miR-24-insensitive CHEK1.Cells were mock transfected or transfected with miR-24 mimic and/orCHEK1 cDNA lacking the 3′UTR. Cell viability was assayed 2 d afterexposure to DNA damage and depicted relative to that of undamaged cells.Immunoblot demonstrates miR-24-mediated decrease in CHEK1 protein andits restoration by transfecting CHEK1 cDNA lacking the 3′UTR.

FIG. 13 shows that miR-24 is upregulated during hematopoietic celldifferentiation into multiple lineages. Heat map for miRNA expression inHL60 and K562 cells differentiated into four different nondividing celllineages, showing single-linkage hierarchical clustering, using Pearsonsquared as a distance metric. miRNA expression in each lane is analyzedrelative to expression in control undifferentiated cells. Thehighlighted cluster shows miRNAs with similar expression profiles. Rangeis from five-fold downregulation (green) to five-fold upregulation(red). Arrows indicate miR-24 cluster miRNAs.

FIG. 14 presents Supplemental Table 1, Primers used for qRT-PCR, andSupplemental Table 2, Mutations introduced in miR-24 binding sites inH2AX-3′UTR.

DEFINITIONS

Cell Proliferative Disorder, Disease, or Condition: The term “cellproliferative disease or condition” is meant to refer to any conditioncharacterized by aberrant cell growth, in some embodiments abnormallyincreased cellular proliferation.

Combination Therapy: The term “combination therapy”, as used herein,refers to those situations in which two or more different pharmaceuticalagents are administered in overlapping regimens so that the subject issimultaneously exposed to both agents.

DNA Damage Agents: The term “DNA damage agents”, as used herein, referto agents that, when applied to cells, damage DNA in the cells. In someembodiments, DNA damage agents are teratogens. As described herein, thepresent invention establishes that presence and/or activity of certainmiRNAs in cells renders those cells hypersensitive to DNA damage agents.Representative such DNA damage agents include, but are not limited to,γ-irradiation, genotoxic drugs, etc.

Dosing Regimen: A “dosing regimen”, as that term is used herein, refersto a set of unit doses (typically more than one) that are administeredindividually separated by periods of time. The recommended set of doses(i.e., amounts, timing, route of administration, etc.) for a particularpharmaceutical agent constitutes its dosing regimen.

Hypersensitive: The term “hypersensitive”, is used herein to refer tocells that are more sensitive than control cells. In some embodiments, acell is considered to be “hypersensitive” as compared with a relevantcontrol if it shows at least about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 25, 40, 25, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000 fold or more susceptible to a particular agent or treatment.

Initiation: As used herein, the term “initiation” when applied to adosing regimen can be used to refer to a first administration of apharmaceutical agent to a subject who has not previously received thepharmaceutical agent. Alternatively or additionally, the term“initiation” can be used to refer to administration of a particular unitdose of a pharmaceutical agent during therapy of a patient.

MicroRNA Agent: A “microRNA agent” as that term is used herein, refersto an entity whose nucleotide sequence is substantially identical tothat of a natural miRNA. As will be appreciated by those of ordinaryskill in the art, naturally-occurring miRNAs are comprised of RNA. Aswill be further appreciated by those of ordinary skill in the art, RNAis a particularly labile chemical. Furthermore, a variety of strategiesare known for preparing molecules that are structural mimics of RNA (andtherefore have a “sequence” in the same sense as RNA) but that may, forexample, have greater stability and/or somewhat altered hybridizationcharacteristics. For example, in some embodiments, such structuralmimics include one or more backbone modifications (e.g., substitution ofphosphorothioate backbone structures for phosphodiester structures foundin RNA) and/or one or more base modifications (e.g., 2′-OMemodifications). In some embodiments, such structural mimics areencompassed within “microRNA agent” as that term is used herein.

Pharmaceutical agent: As used herein, the phrase “pharmaceutical agent”refers to any agent that, when administered to a subject, has atherapeutic effect and/or elicits a desired biological and/orpharmacological effect.

Pharmaceutically acceptable carrier or excipient: As used herein, theterm “pharmaceutically acceptable carrier or excipient” means anon-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type.

Pharmaceutically acceptable ester: As used herein, the term“pharmaceutically acceptable ester” refers to esters which hydrolyze invivo and include those that break down readily in the human body toleave the parent compound or a salt thereof. Suitable ester groupsinclude, for example, those derived from pharmaceutically acceptablealiphatic carboxylic acids, particularly alkanoic, alkenoic,cycloalkanoic and alkanedioic acids, in which each alkyl or alkenylmoiety advantageously has not more than 6 carbon atoms. Examples ofparticular esters include, but are not limited to, formates, acetates,propionates, butyrates, acrylates and ethylsuccinates.

Pharmaceutically acceptable prodrug: The term “pharmaceuticallyacceptable prodrugs” as used herein refers to those prodrugs of thecompounds of the present invention which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humansand lower animals with undue toxicity, irritation, allergic response,and the like, commensurate with a reasonable benefit/risk ratio, andeffective for their intended use, as well as the zwitterionic forms,where possible, of the compounds of the present invention. “Prodrug”, asused herein means a compound which is convertible in vivo by metabolicmeans (e.g. by hydrolysis) to a compound of the invention. Various formsof prodrugs are known in the art, for example, as discussed inBundgaard, (ed.), Design of Prodrugs, Elsevier (1985); Widder, et al.(ed.), Methods in Enzymology, vol. 4, Academic Press (1985);Krogsgaard-Larsen, et al., (ed). “Design and Application of Prodrugs,Textbook of Drug Design and Development, Chapter 5, 113-191 (1991);Bundgaard, et al., Journal of Drug Deliver Reviews, 8:1-38 (1992);Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq. (1988); Higuchiand Stella (eds.) Prodrugs as Novel Drug Delivery Systems, AmericanChemical Society (1975); and Bernard Testa & Joachim Mayer, “HydrolysisIn Drug And Prodrug Metabolism: Chemistry, Biochemistry And Enzymology,”John Wiley and Sons, Ltd. (2002).

Pharmaceutically acceptable salt: As used herein, the term“pharmaceutically acceptable salt” refers to those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, S. M. Berge, et al. describespharmaceutically acceptable salts in detail in J. PharmaceuticalSciences, 66: 1-19 (1977). The salts can be prepared in situ during thefinal isolation and purification of the compounds of the invention, orseparately by reacting the free base function with a suitable organicacid. Examples of pharmaceutically acceptable include, but are notlimited to, nontoxic acid addition salts are salts of an amino groupformed with inorganic acids such as hydrochloric acid, hydrobromic acid,phosphoric acid, sulfuric acid and perchloric acid or with organic acidssuch as acetic acid, maleic acid, tartaric acid, citric acid, succinicacid or malonic acid or by using other methods used in the art such asion exchange. Other pharmaceutically acceptable salts include, but arenot limited to, adipate, alginate, ascorbate, aspartate,benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate,camphorsulfonate, citrate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate,glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate,lauryl sulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and arylsulfonate.

Substantially identical: The term “substantially identical” is used torefer to a nucleic acid sequence that is sufficiently duplicative of areference sequence to share functional attributes of the referencesequence. In particular, a sequence that is “substantially identical” toa reference sequence hybridizes to the complement of the referencesequence. In some embodiments, a sequence is “substantially identical”to a reference sequence if it shows at least 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identity to the reference sequence.In some embodiments, a sequence is “substantially identical” to areference sequence if it shows 100% identity to the reference sequence(i.e., is identical to the reference sequence).

Susceptible to: The term “susceptible to” is used herein to refer to anindividual having higher risk (typically based on geneticpredisposition, environmental factors, personal history, or combinationsthereof) of developing a particular disease or disorder, or symptomsthereof, than is observed in the general population.

Prevention: The term “prevention”, as used herein, refers to a delay inonset and/or a reduction in severity of one or more symptoms orattributes of a disease, disorder or condition, which delay or reductionis observed when a pharmaceutical agent is administered prior to onsetof the symptom(s) or attribute(s).

Therapeutically effective amount: The term “therapeutically effectiveamount” of a pharmaceutical agent or combination of agents is intendedto refer to an amount of agent(s) which confers a therapeutic effect onthe treated subject, at a reasonable benefit/risk ratio applicable toany medical treatment. The therapeutic effect may be objective (i.e.,measurable by some test or marker) or subjective (i.e., subject gives anindication of or feels an effect). A therapeutically effective amount iscommonly administered in a dosing regimen that may comprise multipleunit doses. For any particular pharmaceutical agent, a therapeuticallyeffective amount (and/or an appropriate unit dose within an effectivedosing regimen) may vary, for example, depending on route ofadministration, on combination with other pharmaceutical agents. Also,the specific therapeutically effective amount (and/or unit dose) for anyparticular patient may depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific pharmaceutical agent employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and/orrate of excretion or metabolism of the specific pharmaceutical agentemployed; the duration of the treatment; and like factors as is wellknown in the medical arts.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a pharmaceutical agent thatpartially or completely alleviates, ameliorates, relieves, inhibits,delays onset of, reduces severity of and/or reduces incidence of one ormore symptoms or features of a particular disease, disorder, and/orcondition. Such treatment may be of a subject who does not exhibit signsof the relevant disease, disorder and/or condition and/or of a subjectwho exhibits only early signs of the disease, disorder, and/orcondition. Alternatively or additionally, such treatment may be of asubject who exhibits one or more established signs of the relevantdisease, disorder and/or condition.

Unit dose: The term “unit dose”, as used herein, refers to a discreteadministration of a pharmaceutical agent, typically in the context of adosing regiment.

DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention encompasses the finding that microRNAs (miRNAs)regulate certain key proteins involved in DNA repair and/or cell cycleprogression. In some embodiments, a miRNA modulates levels and/oractivity of one or more DNA repair and/or cell cycle progressionproteins (e.g., in some embodiments, a miRNA suppresses levels and/oractivity of one or more DNA repair and/or cell cycle progressionproteins). In some such embodiments, such modulation renders cellshypersensitive to certain DNA damage agents (e.g., γ-irradiation andgenotoxic drugs, among others).

MicroRNAs

The present invention relates to miRNAs, and particularly to miRNAs thatregulate certain proteins involved in DNA repair and/or cell cycleprogression. In some embodiments, relevant miRNAs are ones whoseexpression level increases or decreases during a particulardevelopmental stage of interest or in response to a particular triggeror event of interest. In some embodiments, relevant miRNAs are oneswhose expression and/or activity levels change during terminaldifferentiation of cells; in some embodiments, relevant miRNAs areup-regulated in terminally-differentiated cells. In some embodiments,relevant miRNAs are up-regulated during terminal differentiation ofhematopoietic cells.

In some embodiments, relevant miRNAs are ones whose expression changesduring a particular developmental stage of interest, or in response to aparticular trigger or event of interest, by an amount that is about 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold ormore.

In some embodiments, relevant miRNAs are ones that regulate cell cycleprogression. In some embodiments, a relevant miRNA suppresses theexpression of cell cycle regulator genes. In some embodiments, arelevant miRNA is characterized in that its overexpression increases thenumber of cells in the G1 phase; in some embodiments, a relevant miRNAis characterized in that its inhibition causes differentiating cells tokeep proliferating.

In some embodiments, a relevant miRNA targets genes that initiatepathways such as synthesis of DNA building blocks; DNA replication; DNAdamage recognition; expression, transcriptional regulation, and/orpost-translational modification of cyclins, cyclin-dependent kinases,and/or other cell cycle regulators. In some embodiments, the miRNAtargets MYC, E2F, and/or their targets.

In some embodiments, a relevant miRNA targets genes that are implicatedin progression through the cell cycle, for example, through G1, the G1/Scheckpoint, S, and/or G2/M. In some embodiments, a relevant miRNAtargets genes that are involved in DNA repair, including for example,genes (e.g., H2AX) that sensitize cells to DNA damaging agents. In someembodiments, a combination of miRNA targets is used. In someembodiments, an miRNA targets a gene that promotes cell proliferation.In some embodiments, an miRNA targets a gene that suppresses cellproliferation (e.g., contributes to blocking cell cycle progression). Insome embodiments, an miRNA targets a gene that participates in DNArepair. In some embodiments, an miRNA targets a gene that suppresses DNArepair.

In some embodiments, a relevant miRNA is selected from the groupconsisting of miR-24 and/or other miRNAs in the same cluster. In someembodiments, a relevant miRNA is miR-22 or miR-125a. For example, insome embodiments, a relevant miRNA is selected from the group consistingof miR-24 (e.g., miR-24-1; miR-24-2), miR-23 (e.g., miR-23a, miR-23B),and miR-27 (e.g., miR-27a, miR-27b), etc. In some embodiments, arelevant miRNA is a member of the let-7 family of miRNAs. In someembodiments, a relevant miRNA is selected from the group consisting ofmiR-48, miR-84, and miR-241. In some embodiments, a relevant miRNA isselected from the group consisting of miR-17, miR-18, miR-19, miR-20,miR-34a, miR-92, miR-125, miR-146a, miR-155, miR-181a, 200a. In someembodiments, a relevant miRNA is one that is found on chromosome 9, oron chromosome 19. In some embodiments, a relevant miRNA is one that isfound in an intergenic region of a chromosome (e.g., chromosome 19). Insome embodiments, a relevant miRNA is a viral miRNA. In someembodiments, a relevant miRNA is a member of the Herpes virus family. Insome embodiments, a relevant miRNA is miR-K12-11. The present Examplesexemplify the invention with respect to miR-24.

Among other things, the present invention provides methods that involveincreasing levels and/or activities of one or more miRNAs, andparticularly of one or more miRNAs that regulates DNA repair and/or cellcycle progression. Some such methods involve increasing levels of anmiRNA agent, in cells. Any of a variety of strategies may be used toincrease levels of an miRNA agent in cells, including, for example,introducing an miRNA agent into cells (e.g., by transfection,transformation, injection, induction, expression from a viral or othervector, etc.).

Alternatively or additionally, the present invention provides methodsthat involve decreasing levels and/or activities of one or more miRNAs,and particularly of one or more miRNAs that regulates DNA repair and/orcell cycle progression. Some such methods involve, for example,providing a competitor agent that competes with a target for interactionwith the miRNA. To give but one specific example, in some embodiments,the present invention provides methods that utilize a “miRNA sponge”that contains multiple copies of an miRNA target sequence. In someembodiments, the present invention provides methods that involveintroducing into a cell an agent that specifically degrades (or targetsfor degradation) a particular miRNA.

DNA Repair Proteins

Cells have evolved the capacity to remove or tolerate lesions in theirDNA (Friedberg, 1985). The most direct mechanisms for repairing DNA arethose that simply reverse damage and restore DNA to its normal structurein a single step. Cells can eliminate three types of DNA damage bychemically reversing it. Such direct reversal mechanisms are specific tothe type of damage incurred and do not involve breakage of thephosphodiester backbone. In some embodiments, DNA damage can lead toformation of thymidine dimers, methylation of guanine bases, methylationof cytosine bases, and/or methylation of adenosine bases, orcombinations thereof. Those of ordinary skill in the art will appreciatethat a variety of agents induce DNA damage (e.g., γ-irradiation and/oradministration of one or more genotoxic drugs, among others). In someembodiments, DNA repair proteins are those that are involved in directreversal of DNA damage (e.g. photolyases and/or methyltransferases). Insome embodiments, DNA repair proteins involved in the direct reversalpathway include human MGMT (Genbank Accession No. M2997 1) and othersimilar proteins.

A more complex mechanism, excision repair, involves incision of the DNAat the lesion site, removal of the damaged or inappropriate base(s), andresynthesis of DNA using the undamaged complementary strand as atemplate. This system of repair can further be categorized into base andnucleotide excision repair.

Base excision repair involves two major classes of repair enzymes,namely, N-glycosylases and AP endonucleases (Wallace, 1988; Sakumi andSekiguchi, 1990; Doetsch and Cunningham; 1990). DNA N-glycosylases areenzymes that hydrolyze the N-glycosidic bond between the damaged baseand the deoxyribose moiety, leaving behind an AP site on the DNAbackbone. AP sites produced by the action of N-glycosylases are actedupon by AP endonucleases, which can make an incision either 3′ to the APsite (class I AP lyase) or 5′ to the AP site (class II AP endonuclease).All those enzymes shown to contain class I AP lyase activity possess anassociated DNA glycosylase activity; however, not all glycosylases areAP lyases. Class II AP endonucleases are the major enzymes responsiblefor the repair of AP sites in DNA.

DNA glycosylases can be defined as enzymes which recognize specific DNAbase modifications and catalyze the hydrolysis of the N-glycosylic bondthat links a base to the deoxyribose-phosphate backbone of DNA (forreview, see Sancar and Sancar, 1988; Wallace, 1988; Sakumi andSekiguchi, 1990). This enzymatic activity results in the generation ofan AP site. To date, several DNA glycosylases have been identified andare classified into two major families: 1) enzymes that possess only DNAglycosylase activity and 2) enzymes that contain both a DNA glycosylaseactivity and an associated class I AP lyase activity; that is, enzymesthat catalyze a beta-elimination cleavage of the phosphodiester bond 3′to an AP site.

In some embodiments, the present invention relates to miRNAs thatregulate certain proteins involved in DNA repair. In some embodiments,relevant DNA repair proteins include those from the base excision repair(BER) pathway, e.g., AP endonucleases such as human APE (NAPE, GenbankAccession No. M80261) and related bacterial or yeast proteins such asAPN-1 (e.g., Genbank Accession No. U33625 and M33667), exonuclease III(ExoIII, xth gene, Genbank Accession No. M22592), exonuclease I (Exol),bacterial endonuclease III (EndoIll, nth gene, Genbank Accession No.J02857), huEndolll (Genbank Accession No. U79718), and endonuclease IV(EndoIV nfo gene Genbank Accession No. M22591). In some embodiments,relevant DNA repair proteins suitable for use in the invention include,additional BER proteins including DNA glycosylases such as,formamidopyrimidine-DNA glycosylase (FPG, Genbank Accession No. X06036),human 3-alkyladenine DNA glycosylase (HAAG, also known as humanmethylpurine-DNA glycosylase (hMPG, Genbank Accession No. M74905), NTG-1(Genbank Accession No. P31378 or 171860), SCR-1 (YAL015C), SCR-2(Genbank Accession No. YOL043C), DNA ligase I (Genbank Accession No.M36067), P-polymerase (Genbank Accession No. M13140 (human)) and8-oxoguanine DNA glycosylase (OGG1 Genbank Accession No. U44855 (yeast);Y13479 (mouse); Y11731 (human)). In some embodiments, relevant DNArepair proteins include histone variants (e.g. H2AX) and transcriptionfactors that regulate expression of DNA repair genes (e.g. XBP1). Insome embodiments, the present invention relates to H2AX.

Cell Cycle Progression Proteins

The sequence of cell cycle events is rigorously controlled at specificcheckpoints to ensure that each discrete stage in the cell cycle hasbeen completed before the next is initiated. Human diseases associatedwith abnormal cell proliferation, including cancer, result when theserigorous controls on cell cycle progression are perturbed. On the otherhand, it is also sometimes desirable to enhance proliferation of cellsin a controlled manner. For example, proliferation of cells is useful inwound healing and where growth of tissue is desirable. Those of ordinaryskill in the art will appreciate that there may be several mechanismsfor cell cycle progression, for example the processes of mitosis and/ormeiosis.

In general, cell cycle progression is regulated by a variety of cellularfactors. For example, two relevant classes of cell cycle progressionregulatory molecules include cyclins and cyclin-dependent kinases(CDKs). In some embodiments, cell cycle progression proteins areselected from the group consisting of cyclin D, cyclin dependent kinase4 (CDK4), retinoblastoma susceptibility protein (RB), E2F, cyclin E,cyclin A, DNA polymerase, thymidine kinase, cyclin dependent kinase 2,cyclin B. In some embodiments, cell cycle progression proteins preventthe progression of the cell cycle. In some embodiments, for example,cell cycle progression proteins are selected from the group consistingof p21, p27, p57, p53, myc, TGFb, p16INK4a, p14arf. In some embodiments,cell cycle progression proteins are involved in DNA replication andrepair checkpoints. In some embodiments, cell cycle progression proteinsare selected from the group consisting of PCNA, CHEK1, BRCA1, FEN1, andUNG.

Applications Cell Proliferative Disorders

In some embodiments, the invention provides methods and reagents fortreating cell proliferative disorders, diseases or conditions. Ingeneral, cell proliferative disorders, diseases or conditions encompassa variety of conditions characterized by aberrant cell growth,preferably abnormally increased cellular proliferation. For example,cell proliferative disorders, diseases, or conditions include, but arenot limited to, atherosclerosis, cancer, immune-mediated responses anddiseases (e.g., transplant rejection, graft vs host disease, immunereaction to gene therapy, autoimmune diseases, pathogen-induced immunedysregulation, etc.), certain circulatory diseases, and certainneurodegenerative diseases.

In certain embodiments, the invention relates to methods and reagentsfor treating cancer. In general, cancer is a group of diseases which arecharacterized by uncontrolled growth and spread of abnormal cells.Examples of such diseases are carcinomas, sarcomas, leukemias, lymphomasand the like. In certain embodiments, the cancer is a hematologicalmalignancy. In certain embodiments, the cancer is a solid tumor. Forexample, in some embodiments, the invention relates to treatment ofrejection following transplantation of synthetic or organic graftingmaterials, cells, organs, or tissue to replace all or part of thefunction of tissues, such as heart, kidney, liver, bone marrow, skin,cornea, vessels, lung, pancreas, intestine, limb, muscle, nerve tissue,duodenum, small-bowel, pancreatic-islet-cell, includingxeno-transplants, etc.; treatment of graft-versus-host disease;autoimmune diseases, such as rheumatoid arthritis, systemic lupuserythematosus, thyroiditis, Hashimoto's thyroiditis, multiple sclerosis,myasthenia gravis, type I diabetes, juvenile-onset or recent-onsetdiabetes mellitus, uveitis, Graves' disease, psoriasis, atopicdermatitis, Crohn's disease, ulcerative colitis, vasculitis,auto-antibody mediated diseases, aplastic anemia, Evan's syndrome,autoimmune hemolytic anemia, and the like; and further to treatment ofinfectious diseases causing aberrant immune response and/or activation,such as traumatic or pathogen induced immune dysregulation.

In some embodiments, the invention relates to treatment of any of avariety of neurodegenerative diseases such as, for example, Alzheimer'sdisease, Parkinson's disease, and/or Huntington's disease.

In some embodiments, the present invention provides methods of treatinga cell proliferative disease, disorder, or condition, by administeringto an individual who is suffering from or susceptible to the cellproliferative disease, disorder, or condition a therapeuticallyeffective amount of an miRNA agent. In some embodiments, thetherapeutically effective amount is an amount sufficient to render cellsof the individual hypersensitive to one or more DNA damage agents. Insome embodiments, the therapeutically effective amount is an amountsufficient to suppress expression and/or activity of one or more DNArepair proteins. In some embodiments, the therapeutically effectiveamount is an amount sufficient to inhibit cell proliferation. In someembodiments, the therapeutically effective amount is an amountsufficient to induce apoptosis.

In some embodiments, a cell is considered to be hypersensitive to one ormore DNA damage agents if it shows increased chromosomal instability(e.g., increased numbers and/or persistence of chromosome breaks),increased cell death rates, and/or increased sensitivity to genotoxicstress.

Increasing Cell Division

In some embodiments, the present invention provides systems forincreasing cell division, for example by modulating (e.g., reducing)levels and/or activities of one or more miRNAs (e.g., miR-24). In someembodiments, for example, modulation of one or more miRNA levels oractivities leads to enhanced division and/or survival as compared withcontrol cells.

In some embodiments, miRNA levels and/or activities are modulatedthrough administration of an agent that modulates (e.g., promotes orsuppresses activity of) the miRNA. To give but one specific example, insome embodiments, level and/or activity of a particular miRNA may bereduced by, for example, administration of an anti-sense agent thathybridizes with the miRNA and competes with one or more natural targetsof the miRNA. In some such embodiments, the anti-sense agent is a miRNAsponge (see above).

In certain embodiments, systems for increasing cell division are useful,for example, in cell culture applications. In some embodiments, any of avariety of cell types are utilized. In some embodiments, stem cell (e.g.embryonic stem cell, hematopoietic stem cell, tissue stem cell, etc)proliferation is increased.

In some embodiments, said systems for increasing cell division areuseful, for example, in the preparation and/or processing of cells ortissues for implantation. For example, in some embodiments, cells arecultured for implantation into a subject (e.g., for tissue replacementand/or repair applications).

In some embodiments, cell proliferation is increased in tissue explants.

Diagnostics

In some embodiments, the present invention provides systems foridentifying cells (and/or individuals) that are suffering from orsusceptible to one or more cell proliferative disorders, for example bydetecting unusual levels or activities of one or more miRNAs and/ortheir targets (whether at the level of RNA or protein). In someembodiments, the targets include one or more DNA repair proteins.

In some embodiments, the present invention provides systems foridentifying cells (and/or individuals) that are hypersensitive to DNAdamage agents, for example by detecting levels or activities of one ormore miRNAs and/or their targets (whether at the level of RNA orprotein). In some embodiments, the targets include one or more DNArepair proteins. In some embodiments, identification of cells (and/orindividuals) that are hypersensitive to DNA damage agents allowsidentification of cells (and/or individuals) who are suffering from orsusceptible to one or more cell proliferative disorders and who arelikely to benefit from therapy that includes administration of one ormore DNA damaging agents or treatments (e.g., γ-irradiation and/oradministration of one or more genotoxic drugs).

Pharmaceutical Compositions

Therapeutic agents (optionally including, for example, miRNA agents) maybe administered to cells or individuals in accordance with the presentinvention, in the context of a pharmaceutical composition. In general, apharmaceutical composition comprises at least one therapeutically activeagent and at least one pharmaceutically acceptable carrier or excipient.Those of ordinary skill in the art will appreciate that atherapeutically active agent may be provided in any of a variety offorms including, for example, in a pharmaceutically acceptable salt orester form.

Representation pharmaceutically acceptable carriers or excipientstypically include, for example, one or more solvents, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants, permeation enhancers, solubilizing agents, and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences, Fifteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1975) discloses various carriers used informulating pharmaceutical compositions and known techniques for thepreparation thereof. Except insofar as any conventional carrier mediumis incompatible with a particular therapeutically active agent, such asby producing any undesirable biological effect or otherwise interactingin a deleterious manner with any other component(s) of thepharmaceutical composition, its use is contemplated to be within thescope of this invention.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, sugars such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose,methylcellulose, ethylcellulose, and cellulose acetate; powderedtragacanth; malt; gelatin; talc; Cremophor (polyethoxylated caster oil);Solutol (poly-oxyethylene esters of 12-hydroxystearic acid); excipientssuch as cocoa butter and suppository waxes; oils such as peanut oil,cottonseed oil; safflower oil; sesame oil; olive oil; corn oil andsoybean oil; glycols; such a propylene glycol; esters such as ethyloleate and ethyl laurate; agar; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives, and antioxidants can also be present inthe composition, according to the judgment of the formulator.

In some embodiments, a pharmaceutically acceptable carrier is selectedfrom the group consisting of sugars such as lactose, glucose andsucrose; starches such as corn starch and potato starch; cellulose andits derivatives such as sodium carboxymethyl cellulose, ethyl celluloseand cellulose acetate; powdered tragacanth; malt; gelatin; talc;excipients such as cocoa butter and suppository waxes; oils such aspeanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, cornoil and soybean oil; glycols such as propylene glycol; esters such asethyl oleate and ethyl laurate; agar; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffersolutions; non-toxic compatible lubricants such as sodium lauryl sulfateand magnesium stearate; coloring agents; releasing agents; coatingagents; sweetening, flavoring and perfuming agents; preservatives andantioxidants; and combinations thereof. In some embodiments, the pH ofthe ultimate pharmaceutical formulation may be adjusted withpharmaceutically acceptable acids, bases or buffers to enhance thestability of the formulated therapeutically active agent or its deliveryform.

Pharmaceutical compositions may be administered in accordance with thepresent invention by any appropriate means including, for example,orally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term parenteralas used herein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques. In many embodiments, pharmaceutical compositionsare administered orally or by injection in accordance with the presentinvention.

Combination Therapy

In accordance with the present invention, two or more therapeuticallyactive agents or regimens may be administered simultaneously to anindividual.

To give but one example, in some embodiments, it may be desirable toadminister an miRNA agent in combination with one or more DNA damageagents or treatments (e.g., γ-irradiation and/or one or more genotoxicdrugs). In some embodiments, combinations of miRNA agents and DNA damageagents or treatments are administered to individuals suffering from orsusceptible to one or more cell proliferation diseases, disorders, orconditions.

EXEMPLIFICATION Materials and Methods Cell Culture and Differentiation

HepG2 cells were grown in DMEM supplemented with 10% FCS. HL60 and K562cells were grown in RPMI-1640 supplemented with 10% FCS. K562 cells(0.5×10⁶ cells/ml) were treated with TPA (16 nM, 2 d) or Hemin (100 μM,4 d) for differentiation into megakaryocytes or erythrocytes,respectively. To induce macrophage or granulocyte differentiation, HL60cells (0.5×10⁶ cells/ml) were treated with TPA (16 nM, 2 d) or DMSO(1.25%, 5 d), respectively. We isolated human polymorphonuclearneutrophils (PMN) from whole blood after removing mononuclear cells andplatelets by Ficoll-Hypaque density gradient centrifugation.Erythrocytes were lysed by treatment with ice-cold isotonic lysis buffer(0.155 M NH4C1, pH 7.4). The remaining PMN cells were washed with Hanks'balanced salt solution and suspended in RPMI medium containing 10% (v/v)FCS. We isolated human macrophages from peripheral blood as described inSong et al. 2003.

RNA Isolation and Quantitative RT-PCR

Total RNA was isolated using Trizol (Invitrogen) and reverse transcribedusing random hexamers and superscript II reverse transcriptase(Invitrogen). qRT-PCR was performed in triplicate samples using the SYBRGreen master mix (Applied Biosystems) and the BioRad iCycler. Primersare provided in Supplemental Table 1. Results were normalized to GAPDH.miRNA quantitative PCR was done in triplicate using the TaqMan MicroRNAAssay from Applied Biosystems as per the manufacturer's instructions andnormalized to U6 SnRNA.

miRNA Microarray

We performed miRNA microarrays as described in Song et al. 2003.

miRNA Mimic and Antisense Oligonucleotide Transfection

HepG2 cells (2.5×10⁵/well) were reverse transfected with 30 nM miRNA tocontrol (cel-miR-67) mimics (Dharmacon) using NeoFx (Ambion) followingthe manufacturer's instructions. K562 cells were transfected with miRNAor control mimics (100 nM) using Amaxa nucleofection following themanufacturer's protocol. K562 cells were treated with TPA (16 nM, 2 d)and were transfected with 100 nM miR-24 ASO using lipofectamine 2000(Invitrogen) and 36 h later these cells were exposed to indicatedconcentrations of bleomycin and cell viability was assessed 2 d later.

Biotin Pull-Down

K562 cells (1×10⁶/well) were transfected with 3′-biotinylated miR-24(Dharmacon) or 3′-biotinylated control miRNA (cel-miR-67) at a finalconcentration of 100 nM in six-well plates in triplicate wells usingAmaxa nucleofection following the manufacturer's protocol. Twenty-fourhours later, the cells were trypsinized and pelleted at 500×g. Afterwashing twice with PBS and resuspension in 0.5 ml lysis buffer (20 mMTris (pH 7.5), 100 mM KCl, 5 mM MgCl₂, 0.3% NP-40, 50 U of RNase OUT(Invitrogen), complete mini-protease inhibitor cocktail (Roche AppliedScience)), and incubation at 4° C. for 5 min, the cytoplasmic extractwas isolated by centrifugation at 10,000×g for 10 min.Streptavidin-coated magnetic beads (50 μl, Invitrogen) were blocked for2 hr at 4° C. in lysis buffer containing 1 mg/ml yeast tRNA and 1 mg/mlBSA (Ambion) and washed twice with 1 ml lysis buffer. Cytoplasmicextract was then added to the beads and incubated for 4 h at 4° C.,following which the beads were washed five times with 1 ml lysis buffer.RNA bound to the beads (pull-down RNA) or from 10% of the extract (inputRNA), was isolated using Trizol LS reagent (Invitrogen). The level ofmRNA in the miR-24 or control pull-down was quantified by qRT-PCR andnormalized to its abundance in the input RNA.

Luciferase Assay

HepG2 cells (2.5×10⁵/well) were reverse transfected in triplicate with30 nM miR-24 mimic, miRNA-328 mimic or control miRNA mimic. Two dayslater, cells were transfected using Lipofectamine 2000 (Invitrogen) withpsiCHECK2 (Promega) vector (0.5 μg/well) containing the 3′UTR of H2AXcloned in the multiple cloning site of Renilla luciferase or control.After 24 hr luciferase activities were measured using the DualLuciferase Assay System (Promega) and TopCount NXT microplate reader(Perkin Elmer) per manufacturer's instructions. Data were normalized toFirefly luciferase. To test whether H2AX mRNA is directly regulated bymiR-24, we cloned the two predicted MREs in the H2AX 3′ UTR into themultiple cloning site of psiCHECK2 and also the mutant versions thatdisrupted base-pairing between the binding sites and miR-24. HepG2 cellswere cotransfected with these plasmids and miR-24 or control mimics for48 h using Lipofectamine 2000, before we performed the luciferase assaysas described above.

Immunoblot

K562 cells (1×10⁶) were transfected with miR-24 mimics or control miRNAmimics (cel-miR-67) as above and 48 h later whole cell lysates wereprepared using RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodiumdeoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0). Protein samples werequantified using Bradford reagent (BioRad) and resolved on 10% SDS-PAGEgels and analyzed by immunoblot probed with antibodies to H2AX (UpstateBiotech), CHEK1 (cell Signaling), histone H3 (Cell Signaling), tubulin(Sigma). All antibodies were used at a dilution of 1:1000.

Chromosome Breakage Analysis

K562 and HepG2 cultures were exposed in duplicate wells to indicateddoses of γ-irradiation and incubated at 37° C. for indicated times in 5%CO₂. Cells were harvested and processed for chromosomal analysisfollowing standard methods (51). 50-75 Wright-stained metaphases foreach condition were scored for chromosomal aberrations.

Single Cell Gel Electrophoresis (Comet) Assay

Single cell comet assays were performed as per manufacturer'sinstructions (Trevigen). Briefly, cells were transfected with siRNAs and60 h later DSBs induced with CPT (2 μM, 1 h, 37° C.). Treated oruntreated cells were collected, resuspended in ice cold PBS at 10⁵cells/ml, mixed with low-melt agarose (1:10 ratio) and spread on frostedglass slides. After the agarose solidified, the slides were successivelyplaced in lysis and alkaline solutions (Trevigen). Slides were thensubjected to electrophoresis (1V/cm distance between electrodes) for 10min in 1×TBE buffer and cells were fixed with 70% ethanol and stainedwith SYBR Green. Nuclei were visualized using epifluorescentillumination on a Zeiss microscope and images analyzed with the NIHImage program. DNA damage was quantified for 75 cells for eachexperimental condition by determining the tail moment, a function ofboth the tail length and intensity of DNA in the tail relative to thetotal DNA, using the software Comet Score (TriTek). Statistical analysiswas by Student's t-test.

Cell Viability Assay

microRNA-transfected K562 or HepG2 cells were seeded (2×10³ cells/100μl) into octuplicate microtiter wells, incubated overnight, and thentreated with indicated reagents or medium for 48 h. Viability wasmeasured by CyQuant Cell Proliferation Assay Kit as per manufacturer'sinstructions (Molecular Probes). Results were expressed as OD₅₂₀relative to that of untreated cells.

Results

Once a cell has terminally differentiated and no longer replicates itsDNA, its need to repair DNA damage is reduced. Although ongoing DNAdamage from oxidative metabolism and exogenous agents may be similar individing and nondividing cells, endogenous double stranded breaks (DSB)that occur during DNA replication and compromise genomic integrity areradically reduced or absent and the danger of propagating damagedchromatin in progeny cells is minimized once a cell has stoppeddividing. Nonetheless, cells that do not divide need to maintain theintegrity of the genes they transcribe. For some long-lived andessentially irreplaceable cells, such as neurons, DNA repair may be moreessential than for short-lived cells, such as terminally differentiatedblood cells. Dividing cells handle the risk of creating DSB during DNAreplication by expressing and activating the homologous recombination(HR) repair machinery in a cell cycle dependent fashion only during Sphase. Moreover, during cell division, DNA damage checkpoint proteinssurvey for unrepaired DNA damage to prevent cell cycle progression atG1/S and G2/M. As a consequence of their reduced needs for DNA repair,nondividing cells have an attenuated response to DSB (1).

The molecular mechanisms behind the down-regulation of DNA repair interminally differentiated cells are generally not well understood. Insome cases, specific repair proteins are down regulated. For instance,Chekl, the orchestrator of cell cycle arrest in response to replicationmediated DNA damage in proliferating cells, is absent in terminallydifferentiated tissues (2) Likewise, E2F1 and p53 expression aredown-regulated in terminally differentiated myotubes (3, 4). mRNA forKu, the DNA binding proteins of the DNA-dependent protein kinase, whichplays a central role in DSB repair by nonhomologous end joining (NHEJ),decreases during differentiation of HL-60 cells into monocytes (5).However, other repair pathways besides DSB repair, such as base excisionrepair (BER) and transcription-coupled repair, which repair lesions ofequal importance in nondividing and dividing cells, may be undiminishedafter terminal differentiation.

The microRNA (miRNA) miR-24 is uniformly up-regulated during terminaldifferentiation of 2 hematopoietic leukemia cell lines, HL60 and K562,into multiple cell lineages as well as in CD8 T cell and muscle celldifferentiation (accompanying manuscript, (6, 7)) (FIG. 4A and FIG. 4B).In the accompanying patent application, U.S. Ser. No. 61/098,696, filedon Sep. 19, 2008, we developed a biochemical approach to identify thegenes regulated by a miRNA by isolating mRNAs that bind to a transfectedbiotinylated mimic of the processed active miRNA transcript. 269 mRNAswere significantly enriched in the miR-24 pull-down. Genes involved inDNA repair and regulating cell cycle progression were highlysignificantly enriched in the pull down.

In particular, of the top 15 enriched gene ontology (GO) processes, 11were involved in various aspects of cell cycle regulation and 2 were inthe response to DNA damage and DNA repair. miR-24 pulled down 31 of 401genes identified as participating in response to DNA damage (7.7%,p=7E-13) and 26 of 313 genes associated with DNA repair (8.3%, p=1E-11).Some of the key nodes in a bioinformatic analysis of the network ofknown direct interactions of the pulled-down gene products includedPCNA, which localizes to DNA replication forks and is required to repairand resolve stalled replication forks (8); CHEK1, the checkpoint proteinthat is activated by ATR and induces cell cycle arrest at G2/M inresponse to unresolved DNA damage; BRCA1, which participates in acomplex that activates DSB repair (9); FEN1, a flap endonuclease whichremoves the 5′ends of Okazaki fragments during lagging-strand DNAsynthesis and participates in BER (10). In addition to these nodes manyof the miR-24-bound genes are key players in DNA repair, including UNG,the major cellular uracil DNA glycosylase (11), EXO1, a 5′-3′exonuclease that has a key role in multiple DNA repair and replicationpathways (12); H2AX, a histone variant that gets phosphorylated at DSBwhere it serves to stabilize checkpoint and repair factors (13); andXBP1, a transcription factor that upregulates DNA repair genes(including FEN 1 and H2AX) (14). The top 3 over-represented GO processesidentified in another data set—the overlap of genes whose mRNAexpression was significantly reduced by miR-24 overexpression with theset of miR-24 predicted targets by TargetScan 4.2—were DNA damagecheckpoint (4 of 44 genes, 9.1%, p=0.0001), DSB repair via HR (3 of 17genes, 17.6%, p=0.0001) and recombinational repair (3 of 17 genes,17.6%, p=0.0001). These analyses strongly suggested that miR-24 mightinhibit DNA repair, with perhaps a special emphasis on genes involved inrepairing lesions that occur during DNA replication.

To identify miRNAs regulating DNA repair during terminal hematopoieticcell differentiation, we analyzed miRNA expression by microarray in twohuman leukemia cell lines—K562 cells differentiated to megakaryocytesusing 12-0-tetradecanoylphorbol-13-acetate (TPA) or to erythrocytes withhemin, and HL60 cells differentiated to macrophages using TPA or tomonocytes using vitamin D3 (FIG. 4). Only a few miRNAs were consistentlyupregulated (by at least 40%) in all four systems of terminaldifferentiation: miR-22, miR-125a and members of the two miR-24clusters—miR-24, miR-23a, miR-23b and miR-27a. miR-24 stood out as themost upregulated miRNA. The only member of the two miR-24 clusters thatwas not consistently upregulated was miR-27b, whose hybridization signalwas substantially lower for all conditions than the other clustermembers, suggesting that hybridization to that probe was inefficient. Wetherefore focused our study on miR-24, which we hypothesized mightregulate terminal differentiation in nondividing cells across multiplecell lineages.

We verified the microarray results by quantitative reverse transcriptionPCR (qRT-PCR). miR-24 was consistently upregulated during terminaldifferentiation of HL60 and K562 cells (FIG. 4A and FIG. 4B) and indifferentiation of CD8 T cells, muscle cells and embryonic stem cells15-17. One of the biggest challenges in studying miRNAs is to identifytarget genes and correlate their downregulation with cellularproperties. Computational algorithms have been developed to predictputative miRNA targets based on complementarity to the 3′ untranslatedregion (UTR) of the target message, particularly of miRNA nucleotides2-8 (the ‘seed’ region)(18). These tools (TargetScan, PicTar, ma22,miRanda) predict overlapping, but distinct, miR-24 target gene sets(18). One strategy to counter this problem is to pursue targetspredicted by multiple algorithms, and with a high prediction score. TheDSB repair gene predicted by all algorithms with a high recognitionscore was H2AFX, encoding histone variant H2AX.

One of the earliest events in the DSB response is phosphorylation ofH2AX at Ser139 by members of the phosphatidylinositol-3 kinase-likefamily of kinases (13). Phosphorylated H2AX (termed γ-H2AX) participatesin DNA repair, replication, and recombination and cell cycle regulation(13). The large domains of γ-H2AX generated at each DSB can bevisualized by immunostaining as nuclear foci. γ-H2AX foci bind andretain an array of cell cycle and DNA repair factors (cohesins, MDC1,Mrel 1, BRCA1, 53BP1, etc.) at the break site (15, 16). Importantly,loss of a single H2AX allele compromises genomic integrity and enhancescancer susceptibility in mice (17, 18). This observation has bothclinical and mechanistic implications. The H2AX dosage effect mayreflect its structural role in chromatin. H2AX comprises −15% ofcellular H2A, and there are two H2A molecules per nucleosome. Thus, H2AXshould be present, on average, in about one of three nucleosomes, andthis density likely is reduced in cells with less H2AX, which mayinterfere with H2AX function. Therefore, a subtle change in cellularH2AX, as might occur with miRNA targeting, may significantly impact DSBrepair. Because of the critical role of H2AX in DNA repair and the knownconsequences of haploinsufficiency, we focused on validating andstudying the effect of miR-24 on H2AX.

H2AX mRNA and protein declined during K562 and HL60 cell differentiation(FIG. 4C to FIG. 4E). The H2AX transcript can be processed alternativelyto a B1.6-kb replication-independent transcript with a poly(A) tail or aB0.6-kb transcript found only in dividing cells, which has a short 3′UTR and lacks a poly(A) tail (19). The shorter transcript, whosesequence is not annotated, might lack miR-24 recognition sites, becausethe H2AX transcript without the 3′ UTR is 505 bases long, leaving onlyabout 100 by for the 3′ UTR. This H2AX transcript containing a shorter3′ UTR and expressed only in dividing cells could be an example of therecently described principle of preferential miRNA regulation of longertranscripts in nondividing cells.

To investigate whether miR-24 regulates H2AX expression, we firstquantified H2AX mRNA in streptavidin pull-downs from K562 cellstransfected for 24 hr with 3′-biotinylated miR-24 or controlbiotinylated miRNA (cel-miR-67) (FIG. 4F) (accompanying patentapplication U.S. Ser. No. 61/098,696, filed on Sep. 19, 2008). Captureof H2AX mRNA in the miR-24 pull-down, analyzed by qRT-PCR normalized toGAPDH, was enhanced by more than 3-fold compared to pull-down with thecontrol miRNA. Pull-down of another housekeeping gene (UBC) did notdiffer from background. Unlike most of the mRNAs pulled-down by miR-24,H2AX is a predicted target of miR-24 by both TargetScan 4.2 and PicTar.Its 3′UTR, which is 1086 nucleotides long, encodes 2 evolutionarilyconserved 7-mer exact matches to the miR-24 seed at positions 88-94 and971-977 and each site has additional pairings to the 3′-region of miR-24(FIG. 4G). PicTar predicts another conserved miRNA interaction (miR-328)with the H2AX 3′UTR.

Next, by qRT-PCR, using primers from the H2AX coding region that measureboth transcripts, we found a four-fold reduction in H2AX mRNA inTPA-treated K562 cells (data not shown). Using primers specific for thelonger transcript, H2AX mRNA declined by two-fold when K562 cells weredifferentiated by TPA to megakaryocytes or by hemin to erythrocytes, andwhen HL60 cells were differentiated by TPA to macrophages or by DMSO togranulocytes (FIG. 4C and FIG. 4D). The level of H2AX protein, measuredafter TPA induction, dropped by 14-fold in K562 cells and 4-fold in HL60cells (FIG. 4E). The strong decrease in H2AX protein levels (relative tothe modest decrease in H2AX mRNA level) during differentiation may beattributed to miR-24—mediated translational inhibition of the residualH2AX transcripts. We first detected increased miR-24 and reduced H2AXmRNA levels in TPA-differentiated K562 and HL60 cells 12 h after addingTPA at which time the cells had stopped dividing (FIG. 1A to FIG. 1D,and FIG. 9B). The relatively high miR-24 and low H2AX mRNA and proteinslevels in vitro differentiated cells were comparable to levels inprimary human peripheral blood monocytes and granulocytes (data notshown). The reduction in H2AX mRNA coincident with increased miR-24 indifferentiated cell lines and primary blood cells could be due to miR-24inhibition of H2AX mRNA expression and/or stability.

We next tested the effect of miR-24 on luciferase expression fromcontrol or H2AX 3′UTR-containing reporter genes in HepG2 cells.Luciferase activity was unchanged from control reporters, but wasreduced more than 2-fold by miR-24 expression (FIG. 4H). miR-24over-expression in HepG2 cells decreased H2AX mRNA by 2-fold, whileprotein expression was reduced even more (˜8-fold) (FIG. 4I and FIG. 4J)Overexpressing miR-328 predicted (by PicTar) to target the 3′UTR of H2AXhad no effect on luciferase activity or H2AX mRNA or protein levels,further underlining the specificity of the miR-24/H2AX interaction (FIG.2A and FIG. 2B). Collectively, these results demonstrate that miR-24binds to the 3′UTR of H2AX mRNA and down-regulates its expression likelyby promoting both mRNA decay and inhibiting translation.

H2AX is a predicted miR-24 target by both TargetScan 4.2 and PicTar. Its3′ UTR, which is 1,086 nucleotides long, encodes two evolutionarilyconserved heptamer exact matches to the miR-24 seed, at positions 88-94and 971-977, and each site has additional pairings to the 3′ region ofmiR-24 (FIG. 4G). PicTar predicts another conserved miRNA interaction(miR-328) with the H2AX 3′ UTR. To identify the miR-24 miRNA recognitionelements (MRE) in the H2AX 3′ UTR, we inserted each of the predictedmiR-24 MREs, as well as MREs with a mutated seed region, into the 3′ UTRof luciferase reporter genes. Luciferase activity was reducedapproximately four-fold when either of the wild-type miR-24 MREs wasinserted, but the mutated MREs (Supplementary Table 2 of FIG. 14) hadlittle effect (FIG. 4K). Therefore, miR-24 regulates H2AX expression bybinding to the two sites predicted by TargetScan and PicTar. AlthoughMRE2 would be found only in the longer H2AX transcript, MRE1 couldpotentially be present in both transcripts. The shorter transcript willneed to be cloned to determine whether this is the case. OverexpressingmiR-328, which is predicted (by PicTar) to target the 3′ UTR of H2AX,had no effect on luciferase activity or H2AX mRNA or protein levels,further underlining the specificity of the miR-24—H2AX interaction (FIG.2A and FIG. 2B). Collectively, these results strongly suggest thatmiR-24 binds to the 3′ UTR of H2AX mRNA and downregulates itsexpression, probably by promoting both mRNA decay and inhibitingtranslation.

To determine whether miR-24-mediated H2AX down-regulation affects DSBrepair, we first evaluated the most serious consequence of unrepairedDSB, chromosomal instability, in K562 cells that were transfected withmiR-24 or mock transfected. The transfection conditions were chosen toachieve a level of H2AX knockdown similar to what is observed during TPAdifferentiation (FIG. 5A). Metaphase spreads were prepared 24 hr afterlow dose γ-irradiation (FIG. 5B). K562 cells over-expressing miR-24 hadtwice as many chromosome breaks and fragments as control cells afterexposure to 0.75 Gy (p<0.001; FIG. 5C, left). SimilarlyTPA-differentiated K562 cells were significantly more sensitive to 0.75Gy radiation than undifferentiated cells (p<0.003; FIG. 5C, middle).Although there were not significantly more breaks 24 hr after exposureto a lower dose of radiation (0.38 Gy), more chromosomal instability wasseen at this dose the next day in miR-24 transfected cells (FIG. 5C,right). Undifferentiated and untransfected K562 cells, which havesignificantly higher endogenous expression of miR-24 and 4-fold lessrelative H2AX mRNA than HepG2 cells, also show more chromosomalaberrations after irradiation than HepG2 cells (FIG. 3).

As another indicator of unrepaired DNA damage, the persistence of DSBwas measured by single cell gel electrophoresis (comet assay) after lowdose bleomycin treatment (FIG. 5D). The comet moment quantifies theextent of unrepaired DNA damage. Although the basal comet moment was notsignificantly changed by miR-24 transfection, the comet tails were5-fold higher (p<0.001) in miR-24 transfected cells compared to controlmiRNA-transfected cells after bleomycin treatment. To determine whetherthe effect of miR-24 on DSB repair was mediated via its effect on H2AX,K562 cells were co-transfected with miR-24 and a miR-24-insensitive H2AXexpression plasmid without the H2AX 3′UTR. The expression plasmid fullyrescued the cells; cells over-expressing miR-24 and H2AX lacking the3′UTR had no significant increase in comet moment after bleomycincompared to cells transfected with the miRNA control and expressionvector. This result strongly suggests that miR-24 regulates DSB repairby controlling H2AX.

Because of impaired DNA damage repair, H2AX deficiency also leads toincreased cell death after exposure to genotoxic drugs. We compared cellviability of K562 cells over-expressing miR-24, miR-328 or mocktransfected after treatment with bleomycin (FIG. 6A, left). Consistentwith the chromosomal breakage and comet assay analysis, cellsover-expressing miR-24 were significantly hypersensitive to DNA damageas were TPA-differentiated cells relative to undifferentiated cells(FIG. 6A, right). miR-328 over-expression, however, had no effect,suggesting that miR-328 is not a physiologically relevant regulator ofH2AX. We also found that unlike miR-24, transfection of miR-328 mimicsdoes not alter H2AX protein levels in K562 cells (FIG. 2B). The effectof miR-24 on DNA damage sensitivity was further confirmed by treatingmiR-24 mimic-transfected HepG2 cells with bleomycin (FIG. 6B, left) andcisplatin (FIG. 6B, right). miR-24 significantly enhanced cytotoxicitycaused by both drugs. The effect of miR-24 on survival was fully rescuedby over-expressing miR-24-insensitive H2AX in both K562 (FIG. 6C) andHepG2 (FIG. 6D) cells. Together these results suggest thatmiR-24-mediated down-regulation of H2AX inhibits the DNA damage responsein terminally differentiated cells.

We next tested the effect of inhibiting miR-24 on sensitivity togenotoxic stress. When K562 cells were transfected with miR-24 antisenseoligonucleotides (ASO), miR-24 expression was reduced even during TPAdifferentiation (FIG. 7A). The reduction in miR-24, which correlatedwith enhanced H2AX mRNA and protein (FIG. 7B), had no effect onundifferentiated K562 cells, but significantly reduced sensitivity tobleomycin in differentiated cells (FIG. 7C).

Why is there a mechanism to dampen DSB repair in terminallydifferentiated cells? One explanation is that most DSBs are generatedduring DNA replication and this mode of regulation allows differentiatedcells to economize and conserve cellular resources under stress-freeconditions. Another possibility is that suppression of repair triggersapoptosis, and this may be preferred to error prone repair via NHEJ (theprimary mode of DSB repair in these cells), which would result inviable, but malfunctioning, cells. Although this solution makes sensefor regenerating cells, such as hematopoietic cells and myocytes, itmight not be a good solution for long-lived terminally differentiatedcells, like neurons, with poor regenerative capacity. It will beworthwhile to determine whether miR-24 is up-regulated during terminaldifferentiation of all cell types or only in lineages that arecontinuously renewing. It is noteworthy that at least one miR-24 clusterhas been reported to be deleted in some poor prognosis cases of CLL(19), a leukemia known to dysregulate key anti-apoptotic genes. Based onour findings here, inappropriate under-expression of miR-24 would bepredicted to enhance DNA repair and thereby enhance resistance tocytotoxic cancer therapies.

This study focused on the effect of miR-24 on H2AX and DSB repair. BothH2AX mRNA and protein are reduced by miR-24 expression. miR-24 is likelyoperating predominantly by inhibiting translation since the effect onprotein levels is much greater than on mRNA. Other proteins recruited toDSB and required for their repair are BRCA1, PCNA and CHEK1, whosetranscripts both precipitate with miR-24 and show proteindown-regulation upon miR-24 over-expression (accompanying patentapplication U.S. Ser. No. 61/098,696, filed on Sep. 19, 2008). BRCA1 andPCNA are important in repairing DNA replication-mediated breaks by HRand CHEK1 arrests dividing cells in response to DNA damage. However,H2AX is required for DSB repair without bias for dividing ornon-dividing cells—it is important for both HR (active only in dividingcells) and NHEJ (throughout the cell cycle) (20). The observation thatDSB repair was completely restored by over-expressing H2AX indifferentiating cells, or cells over-expressing exogenous miR-24,suggests that the key target of miR-24 in DSB repair is H2AX.

REFERENCES

-   Si. P. S. Moorhead, P. C. Nowell, W. J. Mellman, D. M.    Battips, D. A. Hungerford, Exp Cell Res 20, 613 (1960).-   1. T. Nouspikel, P. C. Hanawalt, DNA Repair (Amst) 1, 59 (2002).-   2. C. Lukas et al., Cancer Res 61, 4990 (2001).-   3. P. L. Puri, V. Sartorelli, J Cell Physiol 185, 155 (2000).-   4. L. Belloni et al., Oncogene 25, 3606 (2006).-   5. M. Yaneva, S. Jhiang, Biochim Biophys Acta 1090, 181 (1991).-   6. J. R. Neilson, G. X. Zheng, C. B. Burge, P. A. Sharp, Genes Dev    21, 578 (2007).-   7. Q. Sun et al., Nucleic Acids Res 36, 2690 (2008).-   8. G. L. Moldovan, B. Pfander, S. Jentsch, Cell 129, 665 (2007).-   9. Kramer, J. Lukas, J. Bartek, Cell Cycle 3,1390 (2004).-   10. Y. Liu, H. I. Kao, R. A. Bambara, Annu Rev Biochem 73, 589    (2004).-   11. B. Kavli, M. Otterlei, G. Slupphaug, H. E. Krokan, DNA Repair    (Amst) 6, 505 (2007).-   12. P. T. Tran, N. Erdeniz, L. S. Symington, R. M. Liskay, DNA    Repair (Amst) 3,1549 (2004).-   13. O. Fernandez-Capetillo, A. Lee, M. Nussenzweig, A. Nussenzweig,    DNA Repair (Amst) 3, 959 (2004).-   14. D. Acosta-Alvear et al., Mol Cell 27, 53 (2007).-   15. J. H. Petrini, T. H. Stracker, Trends Cell Biol 13,458 (2003).-   16. M. Stucki, S. P. Jackson, DNA Repair (Amst) 5, 534 (2006).-   17. C. H. Bassing et al., Cell 114, 359 (2003).-   18. Celeste et al., Cell 114, 371 (2003).-   19. G. A. Calin et al., Proc Natl Acad Sci USA 101, 11755 (2004).-   20. M. Shrivastav, L. P. De Haro, J. A. Nickoloff, Cell Res 18, 134    (2008).-   21. Pillai, R. S., Bhattacharyya, S. N. & Filipowicz, W. Trends Cell    Biol. 17, 118-126 (2007).-   22. Ambros, V. Nature 431, 350-355 (2004).-   23. Bartel, D. P. Cell 116, 281-297 (2004).-   24. Sevignani, C., Calin, G. A., Siracusa, L. D. & Croce, C. M.    Mamm. Genome 17, 189-202 (2006).-   25. Tzur, G. et al. PLoS ONE 3, e3726 (2008).-   26. Bartel, D. P. Cell 136, 215-233 (2009).-   27. Mannironi, C., Bonner, W. M. & Hatch, C. L. Nucleic Acids Res.    17, 9113-9126 (1989).-   28. Sandberg, R., Neilson, J. R., Sarma, A., Sharp, P. A. &    Burge, C. B. Science 320, 1643-1647 (2008).-   29. Song, E. et al. J. Virol. 77, 7174-7181 (2003).-   30. Barad, O. et al. Genome Res. 14, 2486-2494 (2004).-   31. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. M. &    Hungerford, D. A. Exp. Cell Res. 20, 613-616 (1960).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention, described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process. Furthermore, it is to be understood that theinvention encompasses all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim. For example, any claim that is dependent on another claim can bemodified to include one or more limitations found in any other claimthat is dependent on the same base claim.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It shouldit be understood that, in general, where the invention, or aspects ofthe invention, is/are referred to as comprising particular elements,features, etc., certain embodiments of the invention or aspects of theinvention consist, or consist essentially of, such elements, features,etc. For purposes of simplicity those embodiments have not beenspecifically set forth in haec verba herein. It is noted that the term“comprising” is intended to be open and permits the inclusion ofadditional elements or steps.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anytargeting moiety, any disease, disorder, and/or condition, any linkingagent, any method of administration, any therapeutic application, etc.)can be excluded from any one or more claims, for any reason, whether ornot related to the existence of prior art.

Publications discussed above and throughout the text are provided solelyfor their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

1. A method comprising steps of: administering to an individual who issuffering from or susceptible to a cell proliferative disorder acomposition comprising an miRNA agent in an amount sufficient to inhibitcell proliferation.
 2. A method comprising steps of: administering to anindividual who is suffering from or susceptible to a cell proliferativedisorder a composition comprising an miRNA agent in an amount sufficientto render cells of the individual hypersensitive to DNA damage agents.3. The method of claim 1, wherein the amount is an amount sufficient tosuppress levels and/or activity of one or more DNA repair proteins. 4.The method of claim 2, wherein the DNA repair proteins are selected fromthe group consisting of photolyases, methyltransferases, APendonucleases, exonucleases (e.g. Exol), histone variants (e.g. H2AX),transcription factors that regulate expression of DNA repair genes (e.g.XBP1), and DNA glycosylases, and combinations thereof.
 5. The method ofclaim 1, wherein the step of administering comprises administering incombination with other cell proliferation disorder treatment methodsand/or therapeutic agents.
 6. A method selected from the groupconsisting of: A method comprising steps of: antagonizing an miRNA agentin a cell, such that cell proliferation is increased; A methodcomprising steps of: administering to an individual who is sufferingfrom or susceptible to a cell proliferative disorder a compositioncomprising an miR-24 agent in an amount sufficient to inhibit cellproliferation in combination with a DNA damage agent and/or cellproliferation disorder treatment method that produces DNA double-strandbreaks, isolating cells from the individual administered the miR-24agent and preparing Wright-stained metaphase chromosome spreads from theisolated cells or staining the isolated cells from the individual withSYBR Green and performing single cell gel electrophoresis on theisolated cells, thereby measuring DNA damage and detecting DNA damagerepair to be reduced in the isolated cells, as compared to controlcells, thereby identifying the individual administered the miR-24 agentto possess reduced DNA damage repair; A method comprising steps of:administering to an individual who is suffering from or susceptible to acell proliferative disorder a composition comprising an miR-24 agent inan amount sufficient to render cells of the individual hypersensitive toa DNA damage agent and/or cell proliferation disorder treatment methodthat produces DNA double-strand breaks, isolating cells from theindividual administered the miR-24 agent and preparing Wright-stainedmetaphase chromosome spreads from the isolated cells or staining theisolated cells from the individual with SYBR Green and performing singlecell gel electrophoresis on the isolated cells, thereby measuring DNAdamage and detecting DNA damage repair to be reduced in the isolatedcells, as compared to control cells, thereby identifying the individualadministered the miR-24 agent to possess reduced DNA damage repair; andA method comprising steps of: administering to an individual who issuffering from or susceptible to a cell proliferative disorder acomposition comprising an miR-24 agent in an amount sufficient todown-regulate the expression of H2AFX, and thereby inhibit cellproliferation, wherein the miR-24 agent is administered in combinationwith a DNA damage agent and/or cell proliferation disorder treatmentmethod that produces DNA double-strand breaks, isolating cells from theindividual administered the miR-24 agent, preparing cDNA from theisolated cells and performing quantitative RT-PCR on the cDNA usingprimers from the H2AX coding region and SYBR Green, thereby measuringH2AFX expression and detecting H2AFX expression to be reduced in theisolated cells, as compared to control cells, thereby identifying theindividual administered the miR-24 agent to possess reduced H2AFXexpression.
 7. The method of claim 1, wherein the miRNA agent isselected from the group consisting of an miR-24 agent (e.g., miR-24-1;miR-24-2), an miR-22 agent, an miR-125a agent, an miR-23 agent (e.g.,miR-23a, miR-23B), an miR-27 agent (e.g., miR-27a, miR-27b), an miR-17agent, an miR-18 agent, an miR-19 agent, an miR-20 agent, an miR-34aagent, an miR-92 agent, an miR-125 agent, an miR-146a agent, an miR-155agent, an miR-181a agent, an 200a agent, an miR-48 agent, an miR-84agent, and an miR-241 agent, and combinations thereof.
 8. The method ofclaim 1, wherein the miRNA agent comprises miR-24.